Type 2 diabetes mellitus (T2DM) is characterized by defects in insulin action and insulin secretion. Hyperglycemia is the primary pathophysiologic abnormality responsible for the microvascular, and to a lesser extent, macrovascular complications. Much in vivo and in vitro evidence in rodents demonstrates that chronic hyperglycemia can induce insulin resistance and cause beta cell dysfunction. However, a rigorous test of the glucotoxicity hypothesis has yet to be performed in man and the basic physiologic/biochemical/molecular mechanisms via which glucotoxicity causes insulin resistance and beta cell dysfunction have yet to be elucidated. In the present grant, we will examine, at the whole body/organ/cellular level, the effect of chronic (72 h), physiologic hyperglycemia on muscle and hepatic insulin sensitivity in NGT individuals without family history of diabetes and in NGT, insulin resistant offspring of two T2DM parents. Using the euglycemic insulin clamp with indirect calorimetry and vastus lateralis muscle biopsies, we will examine the effect of sustained hyperglycemia (+25 mg/dl) on whole body insulin sensitivity, insulin signaling, glycogen synthesis, glucose oxidation, and mitochondria function. We postulate that increased hexosamine flux leads to increased skeletal muscle UDP-N-acetyl glucosamine (UDP-GlcNAc) which subsequently is O-linked to cystosolic proteins causing insulin resistance and mitochondrial dysfunction. We will test this hypothesis directly by quantitating muscle GFAT activity, UDP-GlcNAc levels, total O-GlcNA protein, and O-linked glycogen synthase activity before and after treatment with hexosamidase to remove the UDP-GlcNAc. Similar studies will be performed before and after infusion of glucosamine into NGT family history negative subjects. We also will examine in NGT subjects the effect of chronic (72 h) glucose and glucosamine infusion on hepatic glucose production, hepatic insulin resistance, and gluconeogenesis with the stepped insulin clamp in combination with 3- 3H-glucose, 1-14C-lactate, and D2O. Using the stepped hyperglycemic clamp, we will examine the effect of chronic (72 h) glucose and glucosamine infusion in NGT subjects on beta cell function. Lastly, we will examine the effect of correction of hyperglycemia with an inhibitor of renal glucose transport on muscle/hepatic insulin sensitivity and beta cell function in T2DM subjects. Since SGLT2 inhibitors induce glucosuria and reduce plasma glucose conc without altering basal plasma insulin, FFA, or amino acid levels, this will provide quantitative information about the contributin of glucotoxicity to observed defects in insulin action and insulin secretion in T2DM. These studies have important clinical implications for the treatment of T2DM, since the SLGT2 inhibitors are likely to be the next class of antidiabetic medications approved by the FDA. PUBLIC HEALTH RELEVANCE: T2DM affects ~7% of Americans and is the primary factor responsible for the microvascular, and, to lesser extent, macrovascular complications. Vascular complications are a major source of morbidity/mortality for diabetic patients and represent a major financial expense ($160 billion/year). Delineation of the abnormalities responsible for T2DM will allow development of medications which reverse specific metabolic defects, resulting in decreased incidence of complications and providing more cost effective treatment. Therefore, defining the mechanisms via which glucotoxicity causes insulin resistance and beta cell failure in human T2DM is of paramount importance and will lead to novel, more disease- specific treatments for people with diabetes.